Danièle Godelaine

LAGE-1, a new gene with tumor specificity.

Representational difference analysis was used to identify genes that are expressed in a human melanoma cell line and not in normal skin. A cDNA clone that appeared to be specific for tumors was obtained and the corresponding gene was sequenced. This new gene was named LAGE‐1. Using a LAGE‐1 probe to screen a cDNA library from the same melanoma cell line, we identified a closely related gene, which proved to be identical to NY‐ESO‐1, a gene recently reported to code for an antigen recognized by autologous antibodies in an esophageal squamous cell carcinoma. Gene LAGE‐1 maps to Xq28. It comprises 3 exons. Alternative splicing produces 2 major transcripts encoding polypeptides of 210 and 180 residues, respectively. Expression of LAGE‐1 was observed in 25–50% of tumor samples of melanomas, non‐small‐cell lung carcinomas, bladder, prostate and head and neck cancers. The only normal tissue that expressed the gene was testis. As for MAGE‐A1, expression of LAGE‐1 is induced by deoxy‐azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation. The expression of LAGE‐1 is strongly correlated with that of NY‐ESO‐1. It is also clearly correlated with the expression of MAGE genes. Int. J. Cancer 76:903–908, 1998.© 1998 Wiley‐Liss, Inc.

Cytolytic T-cell responses of cancer patients vaccinated with a MAGE antigen.

Summary: ‘Cancer‐germline’ genes such as the MAGE gene family are expressed in many tumors and in male germline cells but not in normal tissues. They encode shared tumor‐specific antigens, which have been used in therapeutic vaccination trials of metastatic melanoma patients. To establish whether there is a correlation between tumoral regressions and T‐cell responses against the vaccine antigen, we evaluated the responses of patients vaccinated with a MAGE‐3 antigenic peptide or a recombinant virus coding for the peptide. Blood lymphocytes were stimulated with antigenic peptide followed by detection with tetramer, T‐cell cloning, and TCR analysis. In 4/9 regressor patients and in 1/14 progressors we found a low level, usually monoclonal cytolytic T lymphocyte response against the MAGE‐3 peptide.

Phase 1/2 study of subcutaneous and intradermal immunization with a recombinant MAGE-3 protein in patients with detectable metastatic melanoma

The purpose of this phase 1/2 study was to evaluate toxicity, tumor evolution and immunologic response following administration of a fixed dose of a recombinant MAGE‐3 protein by subcutaneous and intradermal routes in the absence of immunologic adjuvant. Thirty‐two patients with detectable metastatic melanoma expressing gene MAGE‐3 were included and 30 received at least one injection with a fixed dose of a ProtD‐MAGE‐3 fusion protein. The immunization schedule included 6 intradermal and subcutaneous injections at 3‐week intervals. Afterward, patients without major tumor progression who required other treatments received additional vaccinations at increasing time intervals. The vaccine was generally well tolerated. Among the 26 patients who received at least 4 vaccinations, we observed 1 partial response and 4 mixed responses. For these 5 responding patients, time to progression varied from 3.5 to 51+ months. An anti‐MAGE‐3 CD4 T‐lymphocyte response was detected in 1 out of the 5 responding patients. The majority of patients had no anti‐MAGE‐3 antibody response. The clinical and immunologic responses generated by the vaccine are rather limited. Nevertheless, given the potential antitumor efficacy and the very mild toxicity of vaccinations, further studies combining MAGE proteins and/or peptides with potent immunologic adjuvants are warranted, not only in metastatic melanoma, but also in the adjuvant setting.

Restoring the Association of the T Cell Receptor with CD8 Reverses Anergy in Human Tumor-Infiltrating Lymphocytes

For several days after antigenic stimulation, human cytolytic T lymphocyte (CTL) clones exhibit a decrease in their effector activity and in their binding to human leukocyte antigen (HLA)-peptide tetramers. We observed that, when in this state, CTLs lose the colocalization of the T cell receptor (TCR) and CD8. Effector function and TCR-CD8 colocalization were restored with galectin disaccharide ligands, suggesting that the binding of TCR to galectin plays a role in the distancing of TCR from CD8. These findings appear to be applicable in vivo, as TCR was observed to be distant from CD8 on human tumor-infiltrating lymphocytes, which were anergic. These lymphocytes recovered effector functions and TCR-CD8 colocalization after ex vivo treatment with galectin disaccharide ligands. The separation of TCR and CD8 molecules could be one major mechanism of anergy in tumors and other chronic stimulation conditions.

Polyclonal CTL Responses Observed in Melanoma Patients Vaccinated with Dendritic Cells Pulsed with a MAGE-3.A1 Peptide

Vaccination with mature, monocyte-derived dendritic cells (DC) pulsed with the MAGE-3168–176 peptide, which is presented by HLA-A1, has been reported to induce tumor regressions and CTL in some advanced stage IV melanoma patients. We present here a precise evaluation of the level of some of these anti-MAGE-3.A1 CTL responses and an analysis of their clonal diversity. Blood lymphocytes were stimulated with the MAGE-3.A1 peptide under limiting dilution conditions and assayed with an A1/MAGE-3 tetramer. This was followed by the cloning of the tetramer-positive cells and by TCR sequence analysis of the CTL clones that lysed targets expressing MAGE-3.A1. We also used direct ex vivo tetramer staining of CD8 cells, sorting, and cloning of the positive cells. In three patients who showed regression of some of their metastases after vaccination, CTL responses were observed with frequencies ranging from 7 × 10−6 to 9 × 10−4 of CD8+ blood T lymphocytes, representing an increase of 20- to 400-fold of the frequencies found before immunization. A fourth patient showed neither tumor regression nor an anti-MAGE-3.A1 CTL response. In each of the responses, several CTL clones were amplified. This polyclonality contrasts with the monoclonality of the CTL responses observed in patients vaccinated with MAGE-3.A1 peptide or with an ALVAC recombinant virus coding for this antigenic peptide.

Vaccination of a Melanoma Patient with Mature Dendritic Cells Pulsed with MAGE-3 Peptides Triggers the Activity of Nonvaccine Anti-Tumor Cells

We previously characterized the CTL response of a melanoma patient who experienced tumor regression following vaccination with an ALVAC virus coding for a MAGE-A3 Ag. Whereas anti-vaccine CTL were rare in the blood and inside metastases of this patient, anti-tumor CTL recognizing other tumor Ags, mainly MAGE-C2, were 100 times more frequent in the blood and considerably enriched in metastases following vaccination. In this study we report the analysis of the CTL response of a second melanoma patient who showed a mixed tumor response after vaccination with dendritic cells pulsed with two MAGE-A3 antigenic peptides presented, respectively, by HLA-A1 and HLA-DP4. Anti-MAGE-3.A1 CD8 and anti-MAGE-3.DP4 CD4 T cells became detectable in the blood after vaccination at a frequency of ∼10−5 among the CD8 or CD4 T cells, respectively, and they were slightly enriched in slowly progressing metastases. Additional anti-tumor CTL were present in the blood at a frequency of 2 × 10−4 among the CD8 T cells and, among these, an anti-MAGE-C2 CTL clone was detected only following vaccination and was enriched by >1,000-fold in metastases relative to the blood. The striking similarity of these results with our previous observations further supports the hypothesis that the induction of a few anti-vaccine T cells may prime or restimulate additional anti-tumor T cell clones that are mainly responsible for the tumor regression.

Monoclonal antibody 57B stains tumor tissues that express gene MAGE-A4

Monoclonal antibody (Mab) 57B, which was raised against a recombinant MAGE‐A3 protein, was tested for its ability to stain cells expressing various members of the MAGE‐A gene family. COS‐7 cells transfected with cDNAs encoding MAGE‐A1, A2, A3, A4, A6, or A12 were stained, whereas those transfected with MAGE‐A8, A9, A10, or A11 cDNAs were not. However, in tissue sections, we observed a different pattern of staining: the antibody effectively stained the tumors that expressed MAGE‐A4 and only these tumors, regardless of the expression of the other MAGE‐A genes. It seems, therefore, that at the level of MAGE gene expression found in tumors, a level clearly lower than that observed in transfected COS cells, only the MAGE‐A4 protein can be reliably detected. We conclude that the 57B Mab should be useful for tumor diagnosis related to therapeutic vaccination involving MAGE‐A4. Int. J. Cancer 86:835–841, 2000.

Functions of Anti-MAGE T-Cells Induced in Melanoma Patients under Different Vaccination Modalities

Tumor regressions have been observed in a small proportion of melanoma patients vaccinated with a MAGE-A3 peptide presented by HLA-A1, administered as peptide, ALVAC canarypox virus containing a MAGE-A3 minigene, or peptide-pulsed dendritic cells (DC). There was a correlation between tumor regression and the detection of anti-MAGE-3.A1 CTL responses. These responses were monoclonal and often of a very low magnitude after vaccination with peptide or ALVAC, and usually polyclonal and of a higher magnitude after DC vaccination. These results suggested that, at least in some patients, surprisingly few anti-MAGE-3.A1 T-cells could initiate a tumor regression process. To understand the role of these T cells, we carried out a functional analysis of anti-MAGE-3.A1 CTL clones derived from vaccinated patients who displayed tumor regression. The functional avidities of these CTL clones, evaluated in lysis assays, were surprisingly low, suggesting that high avidity was not part of the putative capability of these CTL to trigger tumor rejection. Most anti-MAGE-3.A1 CTL clones obtained after DC vaccination, but not after peptide or ALVAC vaccination, produced interleukin 10. Transcript profiling confirmed these results and indicated that approximately 20 genes, including CD40L, prostaglandin D2 synthase, granzyme K, and granzyme H, were highly differentially expressed between the anti-MAGE-3.A1 CTL clones derived from patients vaccinated with either peptide-ALVAC or peptide-pulsed DC. These results indicate that the modality of vaccination with a tumor-specific antigen influences the differentiation pathway of the antivaccine CD8 T-cells, which may have an effect on their capacity to trigger a tumor rejection response.

A reversible functional defect of CD8(+) T lymphocytes involving loss of tetramer labeling

We have observed that human CTL clones lose their specific cytolytic activity and cytokine production under certain stimulation conditions, while retaining an antigen‐dependent growth pattern.These inactive CTL simultaneously lose their labeling by an HLA‐peptide tetramer, even though the amount of TCR‐CD3 at their surface is not reduced. The tetramer‐negative cells recover tetramer staining and cytolytic activity after stimulation with tumor cells in the presence of a supernatant of activated lymphocytes. Our results suggest the existence of a new type of functional defect of CTL. They also indicate that tetramers may fail to reveal some CTL bearing the relevant TCR, even when such functionally arrested CTL retain the potential to participate in immune responses because their defect is reversible.

Identification and characterization of the tumor-specific P1A gene product

Summary— In murine mastocytoma P815, gene P1A directs the expression of antigens P815A and B which are the target of a T cell‐mediated rejection response in syngeneic animals. This gene is expressed at a high level in various tumors, but is silent in normal tissues except testis and placenta; its activation is thus possibly related to malignant transformation. An anti‐synthetic peptide rabbit antiserum reacted by immunoblotting with a cellular protein migrating near 40 kDa on SDS‐PAGE. The immunoreactive protein was detected only in lysates from cells which express antigen P815A: P1.HTR mastocytoma cells and, after transfection with cosmids carrying the P1A gene, the antigen‐loss variant PO.HTR cells and DAP‐3 H‐2L fibroblasts. The identity of this protein as the P1A gene product was confirmed by cell‐free transcription‐translation of the P1A cDNA, the product of which also migrated near 40 kDa in SDS‐PAGE and was captured by protein A‐Sepharose in the presence of the antiserum. Subcellular fractionation by differential and isopycnic centrifugation indicated that the P1A protein is associated with cytoplasmic membranes demonstrating a broad distribution with respect to size and density. Immunofluorescence microscopy also revealed a cytoplasmic signal, particularly intense in small vesicles, which coincides with that produced by an anti‐mouse type I collagen guinea pig antiserum except near the cell periphery where the P1A signal is weaker. We conclude that the P1A protein is bound to membranes of the secretory pathway, at a concentration which goes increasing from the endoplasmic reticulum to secretion vesicles. The N‐terminal portion of the protein was readily removed by proteolytic enzymes in the absence of detergent, suggesting a localization at the cytoplasmic surface. The P1A protein is renewed with a half‐life of 50 mm and is readily phosphorylated upon metabolic labeling of mastocytoma cells with [32P]‐orthophosphate. When immunoisolated from cells lysed with Nonidet P‐40, the P1A protein is accompanied by a 62‐kDa protein which also exists in a phosphorylated form. Thus, it is probably associated with another phosphoprotein, either as a stable functional unit, or as a dissociable complex.